Measurements of the affinity, potency and efficacy of ligands to G protein-coupled receptors (GPCRs) are fundamental to pharmacology and drug discovery. Cellular functional assays measuring the production of GPCR second messengers such as cAMP and inositol phosphates, are used routinely and remain conceptually unchanged since their introduction. However, by their nature 2nd messengers are highly amplified and counter-regulated responses well downstream of the biophysical events resulting from ligand-receptor interactions. Further, dose response curves neglect the underlying kinetics of receptor ligand binding and can therefore give misleading results for both agonist and antagonist potency measurements. The potential for kinetic properties to determine improved clinical utility of GPCR targeted drugs is becoming more broadly appreciated with the characterization of successful therapeutics such as candesartan, maraviroc and others. However, the measurement binding kinetics and intrinsic efficacy in a drug discovery setting is rare due to the lack of appropriate methods. We hypothesize that availability of these properties early in the medicinal chemistry assay cascade for most compounds, rather than retrospectively for a few advanced compounds, will improve the overall efficiency of medicinal chemistry efforts. Recently, GPCR biosensors have been developed to measure binding kinetics and intrinsic efficacy by changes in Fluorescence Resonance Energy Transfer as a function of receptor conformation, independent of other cellular components. We propose to adapt these biosensors for routine use in a drug discovery environment and test the transferability this approache to several additional GPCR's of potential therapeutic interest. This project will define a path by which biosensors can be developed and validated for drug discovery applications and specific sensors will be developed to enable multiple drug discovery programs. Since this is the largest family of validated drug targets, general tools which enhance medicinal chemistry efforts to create efficacious therapeutics are likely to have a significant long term impact on public health. PUBLIC HEALTH RELEVANCE: The goal of this project is to develop GPCR biosensors for use in drug discovery in order to provide a method for routine measurement of ligand binding kinetics and intrinsic efficacy. If successful, this will improve the ability of medicinal chemists to design drugs which act at GPCR targets that may be useful for a wide range of human diseases.